Method of analysis of aldehyde and ketone by mass spectrometry

ABSTRACT

Method of identification and quantitative analysis of aldehyde(s) and/or ketone(s) in a sample by mass spectrometry using stable isotope labeled internal standard is provided. Said internal standard is prepared by reaction of an authentic sample of said aldehyde(s) and/or ketone(s) with a stable isotope labeled reagent, and is added to a sample containing said aldehyde(s) and/or ketone(s). Said aldehyde(s) and/or ketone(s) in said sample is then quantitatively converted to a chemical compound of identical structure, except the stable isotope atoms, as that of said internal standard using a non-labeled reagent. Said sample is then extracted and the extract is analyzed by mass spectrometry. Identification and quantification of said aldehyde(s) and/or ketone(s) are made from a plot of ion ratio of said converted aldehyde and/or ketone to said internal standard versus aldehyde and/or ketone concentration.

CROSS-REFERENCE TO RELATED APPLICATIONS

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STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

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BACKGROUND OF THE INVENTION

This invention pertains to methods of quantitative analysis of aldehydesand ketones in a sample by isotope dilution mass spectrometry. Thestable isotope labeled oximes and hydrazones are used as internalstandards. The sample may be a biological fluid, such as serum, urineetc., or an aqueous sample such as an environmental or an agriculturalsample.

While various methods of analysis such as immunoassays andchromatographic analysis—LC (liquid chromatography), GC (gaschromatography), and TLC (thin layer chromatography)—have been reportedfor identification and determination of levels of aldehydes and ketonesin analytical samples, the absolute and unequivocal identification andquantitative analysis of those compounds are combinations ofchromatographic analysis and MS (mass spectrometry) such as GC-MS andLC-MS. The accuracy and precision of these methods are usually thehighest when stable isotope analogs of the analytes are used as internalstandards. The mass spectrometry method of analysis using stable isotopeinternal standards is commonly called isotope dilution massspectrometry. This method takes advantage of the similar chemical andphysical behaviors of analytes and their respective isotope labeledinternal standards towards all phases of sample preparation and alsotowards instrument responses. It uses the mass differentiation betweenanalytes and their respective internal standard in mass spectrometry forquantification. The requirement for this method of analysis is theavailability of stable isotope labeled internal standards.

The commonly used stable isotope labeled internal standard of an analyteis a chemical compound that has the same chemical structure as that ofthe analyte except that one or more substituent atoms are stableisotopes. Four commonly used stable isotopes are deuterium, carbon-13,nitrogen-15, and oxygen-18. For every hydrogen atom that is replaced bya deuterium atom, the molecular weight of resulting chemical compound isincreased by one mass unit. This is also true for replacing a carbonatom with a carbon-13 atom, or by replacing a nitrogen atom with anitrogen-15 atom. In the case of replacing an oxygen atom with anoxygen-18 atom, the molecular increase is two mass units. Although theacceptable stable isotope labeled internal standard for isotope dilutionmass spectrometry method is the one that is not contaminated with any ofthe unlabeled material, the ideal one should be the one with the highestisotopic purity and contains as many stable isotope atoms as possible.The ideal one, however, must not contain any labeled isotope that can beexchanged for the unlabeled isotope under particular sample preparationconditions.

These criteria of an ideal stable isotope labeled internal standardpresent a challenge for organic synthesis chemists who help theanalytical chemists in the analysis. Most often the synthesis of stableisotope internal standards is not simply an isotope exchange reaction.Easily exchangeable atoms are usually avoided due to possiblere-exchange during sample preparation steps. Organic chemists often haveto carry out multi-step synthesis to make stable isotope labeledinternal standards. Even though many stable isotope labeled reagents arecommercially available, the choice of appropriate labeled reagent forchemical synthesis of stable isotope labeled internal standards is stillvery limited. The limited isotope labeled reagents and the multi-stepsynthesis contribute to the high cost of synthesis of stable isotopeinternal standards. Even if the analytical chemist who carries out theanalysis can afford the cost of the synthesis, there is also a timefactor that he or she has to consider before ordering the synthesis.Situations where organic chemists spent weeks and months on a synthesisproject and came up with nothing at the end were common. This inventionoffers a solution for this problem.

The objective is a short and reliable method of preparing a stableisotope labeled internal standard that is suitable for the analysis ofan analyte in question, but not the synthesis of the stable isotopelabeled analyte. Within the context of the isotope dilution massspectrometry method, both analyte and its internal standard have to haveidentical chemical structures, with the exception of the isotope atomswhich provide the mass differentiation upon mass spectrometric analysis.Analytical chemists who uses GC-MS for their analysis often “derivatize”the analyte and its stable isotope labeled analyte (used as internalstandard) into chemical compounds that can easily pass through the GCcolumn or else provide better instrumental responses. The analysisbecomes the analysis of the “derivatized” analyte and the “derivatized”internal standard, but still provides comparably accurate results ofconcentrations of the analyte itself. Examples of these analyses arefound in cited references. Using similar reasoning, one can synthesize astable isotope derivative of the analyte by reacting it with a stableisotope labeled reagent. The resulting isotope labeled chemical compoundcan be used as internal standard in the analysis of the analyte,providing that the analyte in the analyzed sample will be converted to achemical compound of identical structure as that of the internalstandard using a non-labeled reagent. There are 3 requirements for theusefulness of this method:

1. The analyte in the sample must be quantitatively converted to thecompound of identical structure (except the labeled atoms) as that ofthe added isotope labeled internal standard using a non-labeled reagent.

2. Absolutely no conversion of the isotope labeled internal standard tothe non-labeled compound because the conversion of the analyte happensin the sample in the presence of the added isotope labeled internalstandard.

3. The conversion of the analyte into the compound of identicalstructure as that of the added isotope labeled internal standard has tobe accomplished before any isolation method i.e. extraction, isperformed.

The first two requirements relate to the chemistry of the analyte inquestion. The efficiency of a chosen chemical reaction depends on thetype of reaction which, in turn, depends on the type of functionalgroups of the analyte. This invented method relates to the analysis ofaldehydes and ketones whose chemistry focus on the reactivity of thecarbonyl functional groups of the analyte.

Quantitative reactions of aldehydes and ketones in aqueous samples are:

1. Conversion to an oxime using an alkoxyl amine.

2. Conversion to a hydrazone using an alkyl hydrazine.

There are other reactions of aldehydes and ketones that are veryefficient, but the above conversion reactions are very efficient inaqueous environment and can be performed at room temperature and in arelatively short reaction time. These are necessary and practicalfeatures for routine analysis of aldehydes and ketones in aqueoussamples.

BRIEF SUMMARY OF THE INVENTION

The current invention provides for a method of identification andquantification of aldehyde(s) and/or ketone(s) in a sample by isotopedilution mass spectrometry. The stable isotope labeled internalstandard(s) of said aldehyde(s) and/or ketone(s) is synthesizedbeforehand by reacting a sample containing said analyzed aldehyde(s)and/or ketone(s) with a labeled reagent. Following this step, saidstable isotope labeled internal standard(s) is then added to a samplecontaining said analyzed aldehyde(s) and/or ketone(s). Said analyzedaldehyde(s) and/or ketone(s) is then converted to a non labeledanalog(s) of said labeled internal standard(s) with identical chemicalstructure as said labeled internal standard(s) except for the stableisotope atoms using a non-labeled reagent. Both said converted analyzedaldehyde(s) and/or ketone(s) and its corresponding said stable isotopelabeled internal standard(s) are then extracted and analyzed by massspectrometry. Said stable isotope labeled internal standard(s) providedin the current invention are labeled oxime(s) and hydrazone(s) analogsof said analyzed aldehyde(s) and/or ketone(s). The type of labeledinternal standard(s) used will dictate the labeled reagents used for itssynthesis as well as the non-labeled reagent used to convert theanalyzed aldehyde(s) and/or ketone(s) to the corresponding analog(s).

In comparison with the traditional method of isotope dilution massspectrometric analysis of more than one aldehydes and/or ketones, theinvented method offers the following advantages:

1. The efficiency and simplicity of the above reactions makes possiblethe short, reliable, and quick synthesis of individual stable isotopelabeled internal standards, whereas in the traditional method ofanalysis, stable isotope labeled internal standard of each aldehydeand/or ketone has to be independently synthesized.

2. It is possible to quickly and efficiently synthesize a library ofstable isotope internal standards for the analysis of an entire libraryof aldehydes and/or ketones using these reactions and only onecommercially available stable isotope labeled reagent.

3. Because the synthesis of stable isotope labeled internal standard inthis invented method is usually a one-step synthesis, the entire processof synthesis and sample preparation can be performed in an automatedfashion. The internal standard is prepared in one step, excess isotopereagent is then removed or destroyed, and the prepared internal standardcan be added directly to the samples without purification. Thenon-labeled reagent is added and the sample is ready for extractionshortly thereafter.

These attractive features make the method suitable for high throughputanalysis of aldehydes and/or ketones by isotope dilution massspectrometry.

DETAILED DESCRIPTION OF THE INVENTION

The current invention provides for a method of identification andquantification of aldehyde(s) and/or ketone(s) in a sample by massspectrometry. Said aldehyde(s) and/or ketone(s) has the followingformulas R₁CHO, and R₁R₂CO, wherein R₁ and R₂ are alkyl, aryl, andheteroatom containing cyclic or non-cyclic groups. The current methodcomprises, as an intergral part of the analysis of said aldehyde(s)and/or ketone(s), the following steps:

1. Synthesizing labeled oxime internal standard(s) by reacting anauthentic sample of said aldehyde(s) and/or ketone(s) with a stableisotope labeled reagent to form said oxime internal standard(s) of thegeneral formulas R₁CH═NOR₃ or R₁R₂C═NOR₃, wherein R₃ is a stable isotopelabeled alkyl group. Said R₃ stable isotope labeled alkyl group isselected from the group consisting of CD₃, and CD₂C₆D₅. Said stableisotope labeled reagent is a labeled alkoxyl amine selected from thegroup consisting of labeled methoxylamine and benzyloxyamine.

2. A known amount of said stable isotope labeled oxime internalstandard(s) was then added to said sample containing said aldehyde(s)and/or ketone(s) to be analyzed.

3. Said sample was then contacted with a non-labeled alkoxylamineselected from said group consisting of methoxylamine and benzyloxyamineto quantitatively convert said aldehyde(s) and/or ketone(s) in thesample into said oxime(s) of identical structure as that of said oximeinternal standard(s) mentioned above except for the stable isotopeatoms.

4. Appropriate extraction methods were then used to isolate saidoxime(s) and their corresponding oxime internal standard from saidsample. Concentration of said oxime(s) were determined and quantified bymass spectrometry and based on the ratio of said converted oxime(s) andtheir corresponding oxime internal standard.

In another aspect of the present invention, said labeled internalstandard is a stable isotope labeled hydrazone. In this embodiment, saidstable isotope labeled hydrazone(s) is synthesized by reacting anauthentic sample of said aldehyde(s) and/or ketone(s) with a stableisotope labeled reagent to form said hydrazone internal standard havingthe following formula R₁CH═NNHR₃ or R₁R₂C═NNHR₃ wherein R₃ is a stableisotope labeled alkyl group selected from the group consisting of CD₃,and CD₂C₆D₅. Said stable isotope labeled reagent is a labeled hydrazineselected from a group consisting of labeled methyl hydrazine and labeledbenzyl hydrazine. Also, in this embodiment, said analyzed aldehyde(s)and/or ketone(s) is converted to a hydrazone of identical structure asthat of said hydrazone internal standard except for the stable isotopeatoms by contacting said sample with a non-labeled alkylhydrazineselected from a group consisting of methylhydrazine and benzylhydrazine.

EXAMPLE Analysis of Donepezil in Human Plasma

Step 1: Preparation of Donepezil methoxyloxime-d3.

A solution of 5 mg of Donepezil in 0.5 ml tetrahydrofuran was treatedwith 10 equivalents of hydroxylamine hydrochloride and 0.5 ml 5N sodiumhydroxide. The resulting solution was stirred for 20 hours then thereaction solution was extracted with ethyl acetate-hexane mixture. Thecombined organic extracts were dried with magnesium sulfate andfiltered. The filtered solution was concentrated to give 2 mg crudedonepezil oxime. This crude donepezil oxime was dissolved in 0.5 mltetrahydrofuran and was treated with 1 mg 60% sodium hydride in mineraloil. After 15 minutes of stirring, 3 equivalents of iodomethane-d3 wasadded and the reaction continued to stir for 2 hr. the reaction wasconcentrated and was quenched with 1 ml of water. The quenched reactionwas extracted with ethyl acetate-hexane mixture and the combinedextracts were dried and concentrated. The residue was purified by columnchromatography using silica gel as absorbant and hexane ethyl acetatemixture as eluant. The fractions containing clean Donepezil methoxyloxime-d3 were combined and concentrated to give 0.5 mg product as anoil. MS analysis gave MH+412.

Step 2: Preparation of Working Standard Solutions and Internal StandardSolution.

Working standard solutions of donepezil were prepared by weighingdonepezil and diluting the stock solution to appropriate concentrationas follows: Solution A  2 ng/ml in ethyl acetate B  5 ng/ml C  10 ng/mlD  20 ng/ml E 100 ng/ml

Working quality control standard solutions of donepezil were prepared byindependently weighing donepezil and diluting the stock solution toappropriate concentration as follows QC Solution J  3 ng/ml in ethylacetate K 70 ng/ml

Working internal standard solution of donepezil were prepared bypreparing a stock solution of donepezil methoxyloxime-d3 and dilutingthe stock solution to a working concentration of 10 ng/ml in ethylacetate.

Step 3: Preparation of Calibration Samples and Quality Control Samplesin Human Plasma.

Donepezil-free human plasma aliquots of 0.1 ml were treated with 1000 ulof solution A to G to make calibration samples A to G.

Donepezil-free human plasma aliquots of 0.1 ml were treated with 1000 ulof solution J and K to make quality control samples J and K.

Both calibration samples and quality control samples were then treatedwith 400 ul of the internal standard working solution.

Step 4: Conversion to Oximes and Extraction.

To all prepared samples were added 10 ul of 5N aqueous sodium hydroxidefollowed by 100 ul of a 100 mg/ml solution of methoxylaminehydrochloride in water. The samples were mixed and shaked at roomtemperature for 30 minutes. The samples were extracted with 0.5 ml ethylacetate. Each extract was separated and concentrated. The residue ofeach extract was reconstituted with 100 ul of acetonitrile.

Step 5: Analysis of Reconstituted Extracts by LC/MS/MS.

A total of 7 reconstituted extracts were loaded on a Perkin Elmerautosampler that was connected to a Perkin Elmer LC pump and a PE SciexAPI 365 MS. Each extract was run through an Symmetry C-18 column of 5 umat a rate of 0.3 ml/min of acetonitrile/water 50/50 mixture. The eluatewas directly fed to the MS ion source. MS data were collected for 1.5min per injection.

MS analysis was performed in MRM mode. m/z 409.2>m/z 185.0 was monitoredfor donepezil methoxyloxime while m/z 412.2>m/z 185.0 was monitored fordonepezil methoxyloxime-d3. Collected data were ploted againstconcentration using McQuan 1.5 sofware. Results are tabulated asfollows:

Donepezil

Internal Standard: is

Weighted (1/x*x)

Intercept=3.073

Slope=0.101

Correlation Coeff.=0.999

Use Area Filename Filetype Accuracy Conc. Calc. Conc. Int. Ratio Keto AStandard 100.711 2.000 2.014 3.276 Keto B Standard 98.088 5.000 4.9043.567 Keto C Standard 97.983 10.000 9.798 4.060 Keto D Standard 104.91420.000 20.983 5.186 Keto E Standard 98.304 100.000 98.304 12.971 Keto JQC 95.618 3.000 2.869 3.362 Keto K QC 95.512 70.000 66.859 9.805

References US patent documents 5,559,038 Sep. 24, 1996 J. Fred Kolhouse6,358,996 Mar. 19, 2002 Michael S. Alexander

Other References

Dennis J. Dietzen et al, “Facilitation of thin-layer chromatographicidentification of opiates by derivatization with acetic anhidride ormethoxyamine”, Journal of Analytical Toxicology, September 1995, page299-303, vol. 19.

Kyle R. Gee et al, “Arene chromium and manganese tricarbonyl analogs ofthe PCP receptor ligands5-methyl-10,11-dihydro-5-H-dibenzo[a,d]cyclohepten-5,10-imine (MK-801)and 10,5-(iminomethano)-10-11-dihydro-5H-dibenzo[a,d]cycloheptene”Journal of Organic Chemistry, 1994, p. 1492-1498, vol.59.

Hiroshi Goda et al, “Facile synthesis of 5-substituted2-acetylthiophenes”, Synthesis, 1992, p.849-851.

Arun K. Ghosh et al, “Stereoselective reduction of alpha-hydroxy oximeethers: a convenient route to cis-1,2-amino alcohols”, TetrahedronLetters, 1991, p.711-714, vol.32.

1. A method of identification and quantification of aldehyde(s) and/orketone(s) in a sample comprising the steps of: a) combining a knownamount of an oxime internal standard with said sample comprising saidaldehyde and/or ketone; b) contacting said sample with an alkoxylamineto convert said aldehyde and/or ketone in said sample into an oxime ofidentical structure as that of said oxime internal standard except forthe stable isotope atoms; c) extracting said sample to isolate saidoxime and said oxime internal standard; and d) analyzing said oxime andsaid oxime internal standard by mass spectrometry.
 2. The method ofclaim 1 wherein said mass spectrometric method is the isotope dilutionmass spectrometric method using isotope labeled internal standard. 3.The method of claim 1 wherein said aldehyde and/or ketone is an aldehydeand/or ketone having the following formula R₁CHO or R₁R₂CO wherein R₁and R₂, are alkyl, aryl, and heteroatom containing cyclic or non-cyclicgroups.
 4. The method of claim 1 wherein said oxime internal standard isa stable isotope labeled internal standard.
 5. The method of claim 1wherein said oxime internal standard is synthesized by reacting anauthentic sample of said aldehyde and/or ketone with a stable isotopelabeled reagent to form said oxime internal standard having thefollowing formula R₁CH═NOR₃ or R₁R₂C═NOR₃, wherein R₃ is a stableisotope labeled alkyl group.
 6. The method of claim 5 wherein saidlabeled group R₃ is selected from a group consisting of CD₃ and C₆D₅,formed by reacting said aldehyde and/or ketone with labeled alkoxylamineselected from a group comprising labeled methoxylamine and labeledbenzyloxyamine.
 7. The method of claim 1 wherein said extraction step c)can be any appropriate separating methods such as solid phaseextraction, liquid-liquid extraction or solid supported liquid-liquidextraction.
 8. The method of claim 1 wherein said alkoxylamine isselected from a group consisting of methoxylamine and benzyloxyamine. 9.The method of claim 1 wherein said sample contains either a singularityor a plurality of aldehyde and/or ketone.
 10. The method of claim 1wherein said multiple aldehydes and/or ketones can be converted to saidoximes using a single alkoxylamine.
 11. The method of claim 1 whereinsaid multiple labeled oxime internal standards can be synthesized fromsaid aldehydes and/or ketones using a single labeled alkoxylamine. 12.The method of claim 1 wherein there is no conversion of said stableisotope labeled oxime internal standard to its corresponding non-labeledoxime compound during step b).
 13. The method of claim 1 wherein saidconverting step b) is performed in an aqueous environment.
 14. Themethod of claim 1 wherein said converting step b) is performed beforethe extraction step.
 15. The method of claim 1 wherein said convertingstep b) is quantitative.
 16. A method of identification andquantification of aldehyde(s) and/or ketone(s) in a sample comprisingthe steps of: a) combining a known amount of a hydrazone internalstandard with said sample comprising said aldehyde and/or ketone; b)contacting said sample with an alkylhydrazine to convert said aldehydeand/or ketone in said sample into a hydrazone of identical structure asthat of said hydrazone internal standard except for the stable isotopeatoms; c) extracting said sample to isolate said hydrazone and saidhydrazone internal standard; and d) analyzing said hydrazone and saidhydrazone internal standard by mass spectrometry.
 17. The method ofclaim 16 wherein said mass spectrometric method is the isotope dilutionmass spectrometric method using isotope labeled internal standard. 18.The method of claim 16 wherein said aldehyde and/or ketone is analdehyde and/or ketone having the following formula R₁CHO and R₁R₂COwherein R₁ and R₂ are alkyl, aryl, and heteroatom containing cyclic ornon-cyclic groups.
 19. The method of claim 16 wherein said hydrazoneinternal standard is a stable isotope labeled internal standard.
 20. Themethod of claim 16 wherein said hydrazone internal standard issynthesized by reacting an authentic sample of said aldehyde and/orketone with a stable isotope labeled reagent to form said hydrazoneinternal standard having the following formula R₁CH═NNHR₃ orR₁R₂C═NNHR₃, where R₃ is a stable isotope labeled alkyl group.
 21. Themethod of claim 20 wherein said labeled group R₃ is selected from agroup consisting of CD₃ and C₆D₅, formed by reacting said aldehydeand/or ketone with labeled alkylhydrazine selected from a groupcomprising labeled methylhydrazine and labeled benzylhydrazine.
 22. Themethod of claim 16 wherein said extraction step c) can be anyappropriate separating methods such as solid phase extraction,liquid-liquid extraction or solid supported liquid-liquid extraction.23. The method of claim 16 wherein said alkylhydrazine is selected froma group consisting of methylhydrazine and benzylhydrazine.
 24. Themethod of claim 16 wherein said sample contains either a singularity ora plurality of aldehyde and/or ketone.
 25. The method of claim 16wherein said multiple aldehydes and/or ketones can be converted to saidhydrazones using a single alkylhydrazine.
 26. The method of claim 16wherein said multiple labeled hydrazone internal standards can besynthesized from said aldehydes and/or ketones using a single labeledalkylhydrazine.
 27. The method of claim 16 wherein there is noconversion of said stable isotope labeled hydrazone internal standard toits corresponding non-labeled hydrazone compound during said convertingstep b).
 28. The method of claim 16 wherein said converting step b) isperformed in an aqueous environment.
 29. The method of claim 16 whereinsaid converting step b) is performed before said extraction step. 30.The method of claim 16 wherein said converting step b) is quantitative.